How to lyse adherent cells?

Cell lysis buffer for extracting cytoskeletal proteins:

• 10 mM PBS, pH 7.4
• 1 mM EDTA
• 1% Triton^® X-100

Protocol: Cell lysis, Adherent Cells:

1. Wash attached cells directly in cell culture dish with ice-cold PBS with gentle rocking.
2. Aspirate PBS and add ice-cold lysis buffer. Approximately 1 mL for a 100 mm dish or 150 cm² flask. Or, approximately 5 mL for a 60 mm dish or 75 cm² flask.
3. Incubate for 15 to 20 minutes on ice, and then scrape adherent cells off dish using a rubber spatula.
4. Gently transfer the cell suspension to a pre-cooled micro-centrifuge tube.
5. Agitate for 30 minutes at 4°C
6. Clarify cell lysate by spinning in micro-centrifuge for 10 minutes at 12,000 RPM at 4°C
7. Gently collect the supernatant in a fresh tube and store on ice or frozen at -20°C or -80°C